Persistent infection with Helicobacter pylori is an important risk factor for a number of serious gastric disorders in humans, including the development of gastric ulcer disease or gastric adenocarcinomas. The vacuolating cytotoxin (VacA) is an important virulence factor that mounting evidence indicates is involved in multiple aspects of H. pylori-mediated colonization, persistence, and pathophysiology. The broad, long-term objective of this research program is to identify how the cellular modulating activities of VacA contribute to the pathogenesis strategies of H. pylori within the host. In this application, we propose to explore two important aspects of VacA cellular intoxication. In Specific Aim 1, we propose experiments to investigate the mechanism underlying recent observations that VacA induces caspasedependent programmed cell death in a mitochondrial-dependent manner. Specifically, we will explore the hypothesis that VacA directly elaborates an activity at mitochondria to induce mitochondrial fission as the trigger that ultimately results in programmed cell death. The proposed experiments are important for revealing aspects of an emerging strategy used by several pathogens to directly target mitochondria as a strategy for modulating host cell death. In Specific Aim 2, we will explore the interactions of VacA with plasma membrane sphingomyelin, which we have identified as an important determinant for both conferring cellular sensitivity to VacA as well as binding the toxin to the cell surface. These studies will reveal novel aspects of the mechanisms underlying cellular intoxication, as well as new approaches for blocking cellular intoxication. Because it is estimated that the prevalence of H. pylori infection is nearly 50% worldwide, the importance of developing efficacious vaccines, chemotherapeutics, and diagnostics for H. pylori cannot be overstated. These studies will not only contribute to our understanding of the fundamental mechanisms of VacA-mediated cytotoxicity, but may reveal novel strategies for the prevention or treatment of H. pylori-mediated disease.